Allium fistulosum leaf agglutinin recombinant protein, its encoding polynucleotide, primer and process for preparation thereof

ABSTRACT

Nucleic acid sequence encoding  Allium fistulosum  leaf agglutinin (AFAL) is disclosed. The invention provides  Allium fistulosum  leaf agglutinin (AFAL) recombinant protein, its encoding nucleotides, primers and the process of preparation thereof, said recombinant protein is useful for insect control and haemagglutination activity. AFAL is found more toxic to sap sucking insect pest  Aphis gossypii  (cotton aphid) and  Bemisia tabaci  (whiteflies) as compared to known  Allium sativum  leaf agglutinin. AFAL can be used in the development of transgenic plants for resistance against sap sucking and chewing pests.

PRIORITY APPLICATIONS

This application is a U.S. National Stage Filing under 35 U.S.C. 371from International Application No. PCT/IN2012/000822, filed on 17 Dec.2012, and published as WO2013098852 on 4 Jul. 2013, which claims thebenefit to Indian Application No. 3850/DEL/2011, filed on 28 Dec. 2011;which applications and publication are incorporated herein by referencein their entirety.

FIELD OF THE INVENTION

The invention relates to Allium fistulosum leaf agglutinin (AFAL)recombinant protein, its encoding polynucleotide, primers and process ofpreparation thereof said Allium fistulosum leaf agglutinin (AFAL)protein is useful for insecticidal activity and haemagglutinationactivity. In particular the present invention relates to nucleic acidsequence (afal) encoding for Allium fistulosum leaf agglutinin (AFAL)applicable for haemagglutination and insect control.

BACKGROUND AND PRIOR ART OF THE INVENTION

Plant lectins, also known as “agglutinins”, are heterogeneous group ofcarbohydrate binding proteins, which are able to bind simple sugarsand/or complex carbohydrates reversibly (Van Damme et al. 1998; CRC CritRev Plant Sci 17:575-692). They show a marked heterogeneity with respectto their molecular structures, sugar binding specificity and temporaland spatial regulation. Mannose binding lectins are widely found inhigher plants and play a significant role in defense due to theirability to recognize high-Mannose-type glycans of microbial pathogensand plant predators (Van Damme et al. 1998, 1998, CRC Grit Rev Plant Sci17:575-692; Van Damme et al. 2004 Trends Plant Sci 9:484-9). Mannosebinding lectin from garlic leaf [Allium sativum leaf agglutinin (ASAL)]is a 25 kDa homodimeric protein, structurally and evolutionarily relatedto Galanthus nivalis agglutinin (GNA) (Smeets et al. 1997a, Plant MolBiol 35: 531-535; Van Damme et al. 1992, Eur J Biochem 206:413-420).Some of the biological properties of ASAL are—(1) it readilyagglutinates rabbit erythrocytes but does not affect human erythrocytes(Bandhopadhyay et. al. 2001, Plant Sci. 161:1025-1033; Smeets et al.1997a, Plant Mol Biol 35: 531-535), (2) it has inhibitory effect againstretrovirus (HIV1 and HIV2) induced cytopathicity in MT-4 cells (Smeetset. al. 1997b, Plant Mol Biol 33:223-234) and (3) it is toxic (growthinhibitory) against a spectrum of insects of order Homoptera,Lepidoptera and Coleoptera. Some important pests inhibited by ASAL areaphids [mustard aphid, peach potato aphid, tobacco aphid (Bandhopadhyayet al. 2001, Plant Mol Biol 33:223-234 Hossain et al. 2006, Crop Sci46:2022-2032; Smeets et al. 1997a, Plant Mol Biol 35: 531-535), redcotton bug (Bandhopadhyay et al. 2001, Plant. Mol. Biol. 33:223-234),brown plant hopper, green leaf hopper (Saha et al. 2006, Plant Mol.Biol. 62:735-52) and cotton leaf worm (Sadeghi et al. 2008, TransgenicRes. 17:9-18). Although exact mechanism of insecticidal action oflectins is still not well understood, three different modes of actionhave been proposed—(1) binding of the lectins to the peritrophic matrixof the midgut, inhibiting nutrient absorption (Harper et al 1998, TissueCell 30: 166-176), (2) binding to glycoproteins on epithelial cells ofthe midgut and disrupting tissue integrity (Powell et al. 1998, J InsectPhysiol. 44: 529-539; Sauvion et al. 2004, J Insect Physiol. 50:1137-1150) and (3) binding to carbohydrate moieties of the sensoryreceptors of insect mouth parts, disrupting membrane integrity andinterfering in the food detection ability of insects (Murdock et al.2002, J Agric Food Chem. 50: 6605-6611). All these mechanisms result indecreased ability of insect to ingest food or absorb nutrients, leadingto delayed development and premature death.

U.S. Pat. No. 5,545,820 (Gatehouse, et al., 1996) discloses the use oflectins having specific mannose-binding ability, derived from familyAmaryllidaceae or Alliaceae for the control of insect pests.WO/1992/002139 relates to the use of lectins having specificmannose-binding ability, derived from family Amaryllidaceae or Alliaceaefor the control of insect pests and the development of transgenic plantsexpressing such lectins. U.S. Pat. No. 5,407,454 relates to selectedplant lectins having larvicidal activity against a number of commoninsect pests of agricultural crops. Insect resistance in the transgenicplants is due to insertion of larvicidal lectin gene in all the cells ofthe plants.

U.S. Pat. No. 6,127,532 (Raikhel) refers to transgenic plants containingcDNA encoding Gramineae lectin. Such transgenic plants expressed barleylectin and stored in in the leaves. The transgenic plants, particularlythe leaves exhibit insecticidal and fungicidal′ properties.

Allium fistulosum

Allium fistulosum L. (Welsh onion, Japanese bunching onion) is aperennial onion. Other names that may apply to this plant include greenonion, spring onion, escallion, and salad onion. These names areambiguous, as they may also be used to refer to any young green onionstalk, whether grown from Welsh onions, common bulb onions, or othersimilar members of the genus Allium. The species is very similar intaste and odor to the related bulb onion, Allium cepa, and hybridsbetween the two (tree onions). The Welsh onion, however, does notdevelop bulbs, and possesses hollow leaves (“fistulosum” means “hollow”)and scapes. Large varieties of the Welsh onion resemble the leek, suchas the Japanese ‘negi’, whilst smaller varieties resemble chives. ManyWelsh onions can be multiplied by forming perennial evergreen clumps.Next to culinary use, it is also grown as an ornamental plant.Historically, the Welsh onion was known as the cibol.

The name “Welsh onion” has become a misnomer in modern English, asAllium fistulosum is not indigenous to Wales. “Welsh” preserves theoriginal meaning of the Old English word “welisc”, or Old German“welsche”, meaning “foreign” (compare wal- in “walnut”, of the sameetymological origin). The species originated in Asia, possibly Siberiaor China.

Culinary Use

In the West, the Welsh onion is primarily used as a scallion or saladonion, but is widely used in other parts of the world, particularly EastAsia.

-   Russia: Welsh onion is used in Russia in the spring for adding green    leaves to salads.-   Asia: The Welsh onion is an ingredient in Asian cuisine, especially    in East and Southeast Asia. It is particularly important in China,    Japan, and Korea, hence the other English name for this plant,    ‘Japanese bunching onion’. Bulb onions were introduced to East Asia    in the 19th century, but A. fistulosum remains more popular and    widespread. In Japan, it is used in miso soup, negimaki (beef and    scallion rolls), among others, and it is widely sliced up and used    as a garnish on teriyaki or takoyaki.-   Jamaica: Known as escallion, the Welsh onion is an ingredient in    Jamaican cuisine, in combination with thyme, scotch bonnet pepper,    garlic and allspice (called pimenta). Recipes with escallion    sometimes suggest leek as a substitute in salads. Jamaican dried    spice mixtures using escallion are available commercially. The    Jamaican name is probably a variant of scallion, the term used    loosely for the spring onion and various other plants in the genus    Allium.    Lacking in the Prior Art

Allium lectin disclosed in present invention shows high insecticidalactivity and therefore novel. Homology of the nucleotide sequence withavailable nucleotide sequences in database shows more than 90% homology.

OBJECTIVES OF THE INVENTION

-   -   a) The main objective of the invention is to provide nucleic        acid sequence which encodes for Allium fistulosum leaf        agglutinin    -   b) Another objective of the present invention is to provide gene        specific primers

SEQ ID NO: 5 GSP 1 (5′-ATGGACAGTACTCCATCTCCTAAAC-3′); and SEQ ID NO: 6GSP 2 (5′-TTAGCCCCTTGGCCTCCTGCA-3′),

-   -    useful for amplification of the gene.    -   c) Another objective of the present invention is to provide        agglutinin recombinant protein having high insecticidal activity    -   d) Another objective of the present invention is the application        of Allium fistulosum leaf agglutinin protein for insect control.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to Allium fistulosum leafagglutinin (AFAL) recombinant protein, its encoding nucleotides, primersand the process of preparation thereof, said protein is useful forinsect control and haemagglutination activity.

In an embodiment of present invention, the process of preparation ofAllium fistulosum leaf agglutinin (afal) recombinant protein, useful forinsecticidal activity and haemagglutination activity, comprising offollowing steps—

-   -   a) Extracting of total RNA from the Allium fistulosum leaves    -   b) Synthesizing of cDNA from total RNA extracted from leaves of        Allium fistulosum    -   c) Designing of primers GSP1 and GSP2 from 5′- and 3′-RACE        fragment of c DNA to clone full-length protein encoding DNA,        designing of primers GSP3 and GSP4 for cloning of the DNA        encoding mature polypeptide of AFAL protein,    -   d) Expressing the DNA encoding the mature AFAL in E. coli SUMO        expression vector where SUMO peptide is fused with AFAL at the        N-terminus and expressed in E. coli under T7 promoter to get        desired AFAL recombinant protein.

In another embodiment of the present invention the primers GSP1 and GSP2comprise:

-   -   1. GSP1 represented by SEQ ID NO:5, and    -   2. GSP2 represented by SEQ ID NO:6.

Allium fistulosum leaf agglutinin (AFAL) recombinant protein is 8-10fold more toxic to cotton aphid (Aphis gossypii) and 6-8 fold towhiteflies (Bemisia tabaci) as compared to well-known Allium sativumleaf agglutinin.

According to the invention a DNA fragment of 651 bp was cloned fromtotal cDNA of A. fistulosum leaves, consisted of, 585 bp long openreading frame encoding AFAL precursor protein of 194 amino acid residueswith 28 amino acid long N-terminal signal peptide and 56 amino acid longC-terminal peptide. The amino acid sequences of AFAL are different fromthe other reported Allium lectin sequences. The cloned genomic DNAsequence of afal showed absence of intron.

In another embodiment of the present invention the nucleic acid sequencehaving SEQ ID NO:2, encodes a polypeptide as represented in SEQ ID NO:4,which is a 110 amino acid residue long mature peptide ofAlliumfistulosum leaf agglutinin(AFAL), having a molecular weight of ˜12 kDa.

In yet another embodiment of the present invention the polypeptide hasthe minimum haemagglutination value of in the range of 6-10 ng/ml.

In yet another embodiment of the present invention the polypeptideexhibits insecticidal activity selected from the group comprisinghaemagglutination, insecticidal, antifungal and anti-prolific activity.

In still yet another embodiment of the present invention the polypeptideexhibits haemagglutination activity selected from the group comprisinghaemagglutination, insecticidal, antifungal and anti-prolific activity.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A. Lane 1, Molecular weight markers; Lane 2, Total soluble proteinfrom leaves of A. fistulosum; Lane 3, Unbound total soluble protein;Lane 4, Column wash (before elution); Lanes 5-8, eluted fractions. FIG.1B. Lane 1, Molecular weight markers; Lane 2, Purified AFAL concentratedon 10 kDa cut-off filtration device.

FIG. 2. Peptide mass fingerprinting of AFAL

FIG. 3. V-bottom plate showing haemagglutination of rabbit erythrocytesby AFAL

FIG. 4 Clustalw analysis of AFAL with other Allium lectins. Amino acididentity of AFAL with other mannose binding lectins vary from 67% to75%. Dark area represents mannose binding domain. (SEQ ID NOs: 3 and12-17).

FIG. 5. Lane 1, Molecular weight markers; lane 2. uninduced bacteriallysate; lane 3-5, sample after 1 h, 2 h and 3 h induction; lane 6,supernatant of 3 hr induced culture; lane 7, protein in inclusion.

FIG. 6. Lane 1, molecular weight markers; lane 2, total E. coli proteincontaining SUMO-AFAL; lanes 3-4 represent purified fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

The leaves of Allium fistulosum were collected from the National Bureauof Plant Genetic Resources, Bhowali, Nainital, Uttarakhand, India, andused for the purification of Allium fistulosum leaf agglutinin (AFAL).It was purified on mannose-agarose affinity column, followed by cut-offfiltration device (Example 1, FIGS. 1 & 2). The purified protein wasused for brief characterization.

AFAL shows several times better insecticidal activity against sapsucking pest Aphis gossypii (cotton aphids), Bemisia tabaci (whitefly)as compared to ASAL (Example 3). Large amount of purified protein isrequired for further characterization. The purification of AFAL fromplant leaves in bulk amount is difficult due to unavailability of plantmaterial and very low accumulation of the lectin in leaves. Expressionof protein in a re combinant system is an alternative approach toproduce the desired protein in large amount, which required the cloningof AFAL encoding gene. The gene (afal) was cloned from cDNA preparedfrom total RNA of leaves of A. fistulosum. The cloning was done by RACE(Rapid Amplification of cDNA Ends) using degenerate primers designedfrom the conserved mannose binding domain.

The cloned DNA fragment gene was of 651 bp, consisted of 585 bp openreading frame, 66 bp 5′ untranslated leader sequence. The full-lengthgene encoding AFAL precursor protein of 194 amino acid residues had 28amino acid long N-terminal signal and 56 amino acid long C-terminalpeptide. It contains three mannose binding domains as reported in thecase of other mannose binding lectins (Example 5). The cloned genomicDNA sequence of afal had no intron. The amino acid sequences of AFAL aredifferent from the other reported Allium lectin sequences (Example 5,FIG. 4).

The gene encoding insecticidal protein was cloned in E. coli expressionvector in fusion with SUMO peptide and the recombinant insecticidalprotein was expressed. The protein was purified on Ni-NTA column. Therecombinant protein showed the insecticidal activity against cottonaphids and whiteflies. The gene encoding the mature peptide was clonedin plant expression vector pBI121 under CaMVE35S promoter (Example 6-8).

In the embodiment of the invention, the cloned full-length gene sequenceof Allium fistulosum leaf agglutinin (afal) similar to SEQ ID NO:1. SEQID NO:1 contains DNA sequence which encodes N-terminal signal peptide,mature peptide and C-terminal peptide.

In another embodiment of the invention, nucleotides encoding N-terminalsignal peptide and C-terminal peptide are removed from SEQ ID NO:1, toobtain the mature protein encoding gene sequence, similar to SEQ IDNO:2.

In another embodiment of the invention, the gene sequence of SEQ ID NO:1was translated to obtain the full-length amino acid sequences of AFALsimilar to SEQ ID NO:3.

In yet another embodiment of the invention, the gene sequence of SEQ IDNO:2 was translated to obtain mature AFAL with amino acid sequencesimilar to SEQ ID NO:4.

In yet another embodiment of the invention, the gene encoding the matureAFAL is cloned in E. coli SUMO expression vector where SUMO peptide isfused with AFAL at the N-terminus and expressed in E. coli under T7promoter.

In yet another embodiment of the invention, the AFAL agglutinated rabbiterythrocytes.

In yet another embodiment of the invention, the AFAL was tested againstinsect pests like cotton aphid (Aphis gossypii) and whiteflies (Bemisiatabaci).

In yet another embodiment of the invention, the gene encoding the matureAFAL was cloned in plant expression vector under constitutive promoterCaMV35S and phloem specific promoters CoYMoV, RSS1, RolC etc. andexpressed in transgenic plants for insect control.

(5) Examples Example 1 Isolation and Purification of the Protein

Allium fistulosum leaves were collected from National Bureau of PlantGenetic Resources, Regional Centre, Bhawali, Uttaranchal, India. Theprotein was purified according to the protocol described (Smeets et al.,1997b). The purified protein was further purified on 50 kDa cut-offfiltration device. The purified protein was concentrated on 10 kDacut-off filter and stabilized in PBS for experiments.

Example 2 Peptide Mass Finger Printing

The purified protein was resolved on SDS-PAGE. The protein band wasexcised and digested with trypsin and used for peptide mass fingerprinting. The data was analyzed on MASCOT search. The peptides werefound matching to the mannose binding lectins (FIG. 2).

Example 3 Haemagglutination Assay

Haemagglutination assays with rabbit RBCs were carried out in V-bottomedmicrotitre plates. Total volume of assay was 100 μl, 50 μl aliquot oftwo-fold serially diluted lectin in PBS was mixed with 50 μl 1 of 2%trypsinized rabbit erythrocytes suspension. Microtitre plate wasincubated for 1 hour at room temperature. Agglutination was assessedvisually. Reciprocal of the highest dilution of lectin showingdetectable agglutination was taken as titer of the haemagglutination

TABLE 1 Haemagglutination assay of AFAL and its comparision with ASALS.N Agglutinin Haemagglutination 1 Allium sativum agglutinin (standard)200 ng/ml 2 Allium fistulosum leaf agglutinin  8 ng/ml

Example 4 Insect Bioassay

Insect bioassay was carried out against sap sucking pest, cotton aphid(Aphis gossypii) and whiteflies (Bemisia tabaci). The known amount ofpurified protein was mixed in synthetic diet and insect mortality datawas recorded at different time interval. The data was used for thecalculation of LC₅₀ using probit analysis.

TABLE 2 Insecticidal activity of AFAL purified from leaves of A.fistulosum. Aphids Whiteflies (Aphis gossypii), (Bemisia tabaci), S.NAgglutinin LC₅₀ LC₅₀ 1 Allium sativum   68 μg/ml   76 μg/ml agglutinin(Standard) 2 Galanthus nivalis 51.39 μg/ml 53.39 μg/ml agglutinin 3Allium fistulosum  7.1 μg/ml  8.5 μg/ml agglutinin

Example 5 Gene Cloning and Characterization

cDNA was synthesized following standard protocol. The 3′ RACE wasperformed with degenerate primer {5′ATGCA(A/G)(C/G)A (G/T)GACTGCAACC-3′;SEQ ID NO:11}(primer sequence was derived from the mannose-binding site,QXDXNXVXY (SEQ ID NO:10), conserved among most of the monocotmannose-binding lectins) and universal primer. For 5′ RACE, RNA wasreversely transcribed with the 5′-RACE CDS Primer. Based on the 3′ and5′ RACE results, primers were designed for amplification of full lengthgene, GSP1 (5′-ATGGACAGTACTCCATCTCCTAAAC-3′; SEQ ID NO:5) GSP2 (5′-GCCCCTTGGCCTCCTGCA-3′; SEQ ID NO:9). The full-length gene was amplifiedand cloned. The mature AFAL encoding DNA was amplified with primers

SEQ ID NO: 7 GSP 3 (5′-AGAAACGTATTGGTGAACAACG-3′); and SEQ ID NO: 8GSP 4 (5′-TTATCTTCTGTAGGTACCAGTAGAC-3′).

The amino acid sequence of AFAL was deduced with Expasy translate tool.The analysis and comparison of the deduced amino acid sequences andnucleotide sequences obtained in RACE was performed with blast p(Standard Protein-Protein BLAST), blastn (Standard Nucleotide-NucleotideBLAST) on NCBI (www.ncbi.nlm.nih.gov) and clustal W.

SEQ ID NO:1 Nucleic acid sequences encoding full-length AFAL

SEQ ID NO:2 Nucleic acid sequences encoding mature AFAL.

SEQ ID NO:3 Amino acid sequence of the Allium fistulosum leaf agglutinin

SEQ ID NO:4 Amino acid sequence of the mature Allium fistulosum leafagglutinin

Nucleic acid sequences encoding full length AFAL SEQ ID NO: 1ATGGACAGTA CTCCATCTCC TAAACTAATG AGCATGACCA 60 CTGTAGCCAC CATCCTAACCATTTTGGCAT CTACATGCAT GGCCAGAAAC GTATTGGTGA 120 ACAACGAAGG ACTGTACGCAGGCCAATCCC TAGTCGTAGA ACAGTACACT TTTACAATGC 180 AGGATGACTG CAACCTTGTACTCTACGAAT ACTGCGCCCC AATCTGGGCC TCAAACACGG 240 GCGTCACCGG CAAAAATGGGTGCAGGGCCG TGATGCAGGC TGATGGCAAC TTTGTGGTCT 300 ACGATGTTAA CGGGCGTGCCGTCTGGGCCA GTAACAGCAG AAGAGGGAAC GGAAACTATA 360 TCCTGGTGCT TCAGGAGGACAGGAACGTTG TTATTTACGG ATCTGATATT TGGTCTACTG 420 GTACGTACAG AAGAGGGCCCGGTCCTGGTC CTGGTGCCGC CTGCAAGTGC GATGACGATG 480 GTCCTGACAT TCGCAGTGCTACTTTGACAG GCACTGTCGA TTTGGGAAGC TGCAACGAGG 540 GATGGGAGAA GTGCGCATCTTTCTACACCA TCCTCGCGGA TTGCTGCAGG AGGCCAAGGG 585 GCTAANucleic acid sequences encoding mature AFAL. SEQ ID NO: 2AGAAACGTAT TGGTGAACAA CGAAGGACTG TACGCAGGCC 60 AATCCCTAGT CGTAGAACAGTACACTTTTA CAATGCAGGA TGACTGCAAC CTTGTACTCT 120 ACGAATACTG CGCCCCAATCTGGGCCTCAA ACACGGGCGT CACCGGCAAA AATGGGTGCA 180 GGGCCGTGAT GCAGGCTGATGGCAACTTTG TGGTCTACGA TGTTAACGGG CGTGCCGTCT 240 GGGCCAGTAA CAGCAGAAGAGGGAACGCAA ACTATATCCT GGTGCTTCAG GAGGACAGGA 300 ACGTTGTTAT TTACGGATCTGATATTTGGT CTACTGGTAC GTACAGAAGA 330 Amino acid sequence of AFALSEQ ID NO: 3 MET ASP SER THR PRO SER PRO LYS LEU MET SER 20MET THR THR VAL ALA THR ILE LEU THRILE LEU ALA SER THR CYS MET ALA ARG ASN VAL 40LEU VAL ASN ASN GLU GLY LEU TYR ALAGLY GLN SER LEU VAL VAL GLU GLN TYR THR PHE 60THR MET GLN ASP ASP CYS ASN LEU VALLEU TYR GLU TYR CYS ALA PRO ILE TRP ALA SER 80ASN THR GLY VAL THR GLY LYS ASN GLYCYS ARG ALA VAL MET GLN ALA ASP GLY ASN PHE 100VAL VAL TYR ASP VAL ASN GLY ARG ALAVAL TRP ALA SER ASN SER ARG ARG GLY ASN GLY 120ASN TYR ILE LEU VAL LEU GLN GLU ASPARG ASN VAL VAL ILE TYR GLY SER ASP ILE TRP 140SER THR GLY THE TYR ARG ARG GLY PROGLY PRO GLY PRO GLY ALA ALA CYS LYS CYS ASP 160ASP ASP GLY PRO ASP ILE ARG SER ALATHR LEU THR GLY THE VAL ASP LEU GLY SER CYS 180ASN GLU GLY TRP GLU LYS CYS ALA SERPHE TYR THR ILE LEU ALA ASP CYS CYS ARG ARG 194 PRO ARG GLYAmino acid sequence of the mature AFAL SEQ ID NO: 4ARG ASN VAL LEU VAL ASN ASN GLU GLY LEU TYR 20ALA GLY GLN SER LEU VAL VAL GLU GLNTYR THR PHE THR MET GLN ASP ASP CYS ASN LEU 40VAL LEO TYR GLU TYR CYS ALA PRO ILETRP ALA SER ASN THR GLY VAL THR GLY LYS ASN 60GLY CYS ARG ALA VAL MET GLN ALA ASPGLY ASN PHE VAL VAL TYR ASP VAL ASN GLY ARG 80ALA VAL TRP ALA SER ASN SER ARG ARGGLY ASN GLY ASN TYR ILE LEU VAL LEU GLN GLU 100ASP ARG ASN VAL VAL ILE TYR GLY SERASP ILE TRP SER THR GLY THR TYR ARG ARG 110 Primer sequence GSP1:SEQ ID NO: 5 (5′-ATGGACAGTACTCCATCTCCTAAAC-3′). Primer sequence GSP2:SEQ ID NO: 6 (5′-GCCCCTTGGCCTCCTGCA-3′). Primer sequence GSP3:SEQ ID NO: 7 (5′-AGAAACGTATTGGTGAACAACG-3′). Primer sequence GSP4:SEQ ID NO: 8 (5′-TTATCTTCTGTAGGTACCAGTAGAC-3′)..

Example 6 Expression of AFAL with N-Terminal Fusion of SUMO in E. coliand Purification of SUMO-AFAL

The gene encoding mature AFAL was cloned in E. coli expression vector infusion with SUMO peptide under T7 promoter. SUMO had (His₆) tag attachedwhich helped in the purification of recombinantly expressed protein onNi-NTA resin. SUMO-AFAL was expressed after induction with IPTG. Theexpression of the recombinant protein was observed every hour for 3hours. After 3 hours of induction, cells were harvested bycentrifugation; suspended in 20 mM TrisCl (pH 8). Bacterial cells werelysed by lysozymen and disrupted by sonication. The lysed bacterialcells were spun and supernatant and pellet were collected andelectrophorased on denaturing PAGE (FIG. 5). Approximately half of therecombinant protein was in the soluble form and rest as inclusion in thepellet.

Purification of SUMO-AFAL from E. coli

Recombinantly expressed SUMO-AFAL was purified on metal-affinity column.Total bacterial protein was loaded on Ni-column, pre-equilibrated withthe buffer (20 mM Tris pH 8, 300 mM NaCl and 10 mM Imidazole). NaCl andImidazole were used to prevent the binding of non-specific proteins tothe column. The column was washed with same buffer having 20 mMimidazole to remove low affinity bound proteins. Finally, the proteinwas eluted with 200 mM Imidazole (FIG. 6).

Example 7 Insect Bioassay with Recombinant Fusion Protein

Insect bioassay was carried out against sap sucking pest, cotton aphid(Aphis gossypii) and whiteflies (Bemisia tabaci). The known amount ofSUMO-AFAL was mixed in synthetic diet and insect mortality data wasrecorded at different time interval. The data was used for thecalculation of LC₅₀ using probit analysis. SUMO-ASAL served as positivecontrol. The results of insect bioassay is shown in the table 3

TABLE 3 LC₅₀ Whiteflies S.N Agglutinin Aphids (Aphis gossypii) (Bemisiatabaci) 1 Recombinant ASAL 55.05 μg/ml 51.2 μg/ml (SUMO-ASAL) 2Recombinant AFAL  4.97 μg/ml 8.80 μg/ml (SUMO-AFAL)Advantages of the Invention

The lectin protein being disclosed in the present invention (AFAL) is6-10 folds more toxic to insects like aphids and whiteflies as comparedto the standard Allium sativum leaf lectin (ASAL). AFAL also showed 25folds higher haemagglutination activity as compared to ASAL. AFAL bindsto lesser number of carbohydrate residues on glycans array as comparedto ASAL. This assures fewer non-specific binding of AFAL tocarbohydrates and therefore expected to be safe as compared to ASAL.

REFERENCES CITED

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The invention claimed is:
 1. A process for preparation of Allium fistulosum leaf agglutinin (AFAL) recombinant protein by amplifying the AFAL gene, wherein the process comprises: a) extracting total RNA from Allium fistulosum leaves; b) synthesizing cDNA from total RNA extracted from the leaves of Allium fistulosum; c) employing primers with a nucleotide sequence as set forth in SEQ ID NO:5 and SEQ ID NO:6 with cDNA of (b) to amplify and obtain a nucleotide fragment encoding full-length AFAL protein; d) employing primers having nucleotide sequences as set forth in SEQ ID NO:7 and SEQ ID NO:8 with said nucleotide fragment to amplify and obtain a polynucleotide fragment encoding mature AFAL protein; e) obtaining an expression cassette comprising said polynucleotide fragment and a promoter; f) introducing said expression cassette in a host to obtain recombinant host cells; and g) culturing said recombinant host cells to obtain recombinant AFAL protein.
 2. Nucleic acid sequence represented by SEQ ID NO:1, obtained by the process claimed in claim 1, wherein SEQ ID NO:1 is comprised of 1 to 585 nucleotides or the sequence complementary thereto, which encodes full-length Allium fistulosum leaf agglutinin polypeptide, wherein nucleotide sequence 583 to 585 is a stop codon.
 3. A cDNA fragment having a nucleic acid sequence as set forth in SEQ ID NO:2.
 4. A method to express Allium fistulosum leaf agglutinin (AFAL) polypeptide, comprising: introducing to a cell genome a vector comprising a nucleotide sequence having SEQ ID NO:1 or SEQ ID NO:2 operably linked to a promoter expressed in E. coli, Pseudomonas, Pichia pastoris or Saccharomyces cerevisiae; and isolating from the cell a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4, wherein the said polypeptide is a 110 amino acid residue long mature peptide of Allium fistulosum leaf agglutinin (AFAL) recombinant protein, having a molecular weight of about 12 kDa.
 5. A method for making a plant resistant to insects, said method comprising producing an amount of isolated polypeptide having an amino acid sequence as set forth in SEQ ID NO: 4 in said plant, effective to exhibit insecticidal activity against the insect, wherein said plant is transformed with a nucleic acid having SEQ ID NO:
 1. 6. The method as claimed in claim 5, wherein the isolated protein is in an amount that exhibits insecticidal activity against Aphis gossypii, Bemisia tabaci, Helicoverpa armigera and Spodoptera litura.
 7. The method as claimed in claim 5, wherein the insect belongs to order lepidoptera, homoptera, coleopteran, or diptera. 